The goal of this research is to determine how the disassembly, turnover, and resculpting of cellular actin filament arrays is regulated by the concerted actions of GMF, Coronin, Srv2/CAP, Twinfilin, Abp1, Cofilin, and AIP1. Each of these core components of the eukaryotic actin machinery is conserved from yeast to humans and has been genetically implicated in promoting actin turnover and remodeling. However, the specific roles and mechanisms of each protein and how they function in groups are only beginning to be understood. The proposed research will elucidate how these factors interact and work together in novel multi-component mechanisms to control actin filament debranching, coalescence, severing, capping, and depolymerization. This will define the molecular basis of diverse biological processes that rely on characteristic actin network geometries and turnover rates, including cell motility, endocytosis, and intracellular transport of vesicles, organelles, and pathogens. To achieve these goals, we will combine genetics, biochemical assays, in vitro multi-wavelength single molecule TIRF microscopy, and live cell imaging approaches. Mechanisms elucidated at the single molecule level in vitro will be tested in vivo using separation-of-function alleles for their importance in diverse biological processes. These include: yeast endocytosis and endosomal fusion with vacuoles, yeast actin cable turnover mediating intracellular transport, actin-based motility of Listeria in infected mammalian cells, and turnover of filopodia, stress fibers, and leading edge networks in mammalian cells. The Aims are: (1) Test the hypothesis that actin filament networks are collaboratively debranched by mechanisms involving GMF, Coronin, Cofilin, and Srv2/CAP. (2) Test the hypothesis that Srv2/CAP and Abp1 interact to form a megadalton-sized complex that remodels actin networks by a novel mechanism of filament sliding and coalescence into bundles. (3) Test the hypothesis that novel mechanisms of actin filament end depolymerization mediated by Twinfilin and Srv2/CAP control the disassembly and length of cellular actin structures. (4) Test the hypothesis that differences in actin filament decoration and spatial organization govern how actin networks are remodeled and turned over.